Cytoplasmic Pink1 activity protects neurons from dopaminergic neurotoxin MPTP

Abstract

PTEN-induced putative kinase 1 (Pink1) is a recently identified gene linked to a recessive form of familial Parkinson's disease (PD). The kinase contains a mitochondrial localization sequence and is reported to reside, at least in part, in mitochondria. However, neither the manner by which the loss of Pink1 contributes to dopamine neuron loss nor its impact on mitochondrial function and relevance to death is clear. Here, we report that depletion of Pink1 by RNAi increased neuronal toxicity induced by MPP(+). Moreover, wild-type Pink1, but not the G309D mutant linked to familial PD or an engineered kinase-dead mutant K219M, protects neurons against MPTP both in vitro and in vivo. Intriguingly, a mutant that contains a deletion of the putative mitochondrial-targeting motif was targeted to the cytoplasm but still provided protection against 1-methyl-4-phenylpyridine (MPP(+))/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. In addition, we also show that endogenous Pink1 is localized to cytosolic as well as mitochondrial fractions. Thus, our findings indicate that Pink1 plays a functional role in the survival of neurons and that cytoplasmic targets, in addition to its other actions in the mitochondria, may be important for this protective effect.

Fig. 1.

Knockdown of Pink1 in NIH 3T3 cell lines or cortical neurons. (A) NIH 3T3 cells were transfected with the siRNA targeting the mouse Pink1 gene, and 24 h after the transfection cells were collected to isolate the RNA; RT-PCR was carried out by using Pink1 primers and S12 primer as an internal control. (B) Alternatively, cortical neurons were infected with adenovirus harboring shRNA Pink1 at the time of plating, and cells were processed for RT-PCR. (C) Cortical neurons after infection with shRNA Pink1 virus or control were processed for immunostaining with Pink1 antibody. (D) Quantification of fluorescent intensity of Pink1-immunostained neurons infected with control and shRNA Pink1 virus. The asterisk denotes significance (P < 0.05). Values are mean ± SEM (n = 30–34). (E) Pink1 knockdown by shRNA Pink1 in cortical neurons sensitizes the cells to MPP⁺. Cortical neurons are transfected with shRNA plasmid 3 days after plating. Cells are treated with 20 μM MPP⁺ for 24 h, and GFP-positive cells are counted based on nuclear integrity. The asterisk denotes significance (P < 0.05) when compared with other conditions.

Fig. 2.

Primary cortical neurons were cultured for 3 days and then transfected with different constructs by calcium phosphate. (A) Cells were treated with 20 μM MPP⁺ for 48 h, and neuronal survival was evaluated by assessing the nuclear integrity of GFP-positive neurons. (B) Neuroprotective effect of WT Pink1 or ΔNPink1 (N-terminal truncated Pink1) in cortical neurons against MPP⁺. The mutant G309D or K219M did not show any protection. The data are the mean ± SEM (n = 3). The asterisk denotes significance (P < 0.05) when compared with MPP⁺ control.

Fig. 3.

Immunostaining of COS-7 cell lines with WT Pink1 or ΔNPink1. (A) Cells are transfected with WT or ΔNPink1, and, 24 h after transfection, cells are incubated with mitotracker and fixed for immunostaining. The cells stained with anti-Flag antibody to visualize Pink1 and images are taken by confocal microscopy. (B) NIH 3T3 cells are transfected with GFP, WT, ΔNPink1, and K219M Pink1 construct. Then, 24 h after transfection, cells are fractionated into cytosolic and mitochondrial fraction with an equal volume of buffer. Electrophoresis was carried out with an equal volume from each fraction.

Fig. 4.

WT or ΔNPink1 provides protection against MPTP administration. The adenoviruses (2 μl, 1 × 10⁷ particles per μl) expressing WT, ΔNPink1, G309D, and K219M Pink1 were injected directly into the striatum of animals 7 days before the initiation of MPTP treatment. A GFP-expressing virus was used as a control. Brains were sectioned into 14-μm slices for TH staining. (A) Representative images of TH-immunopositive neurons of the ipsilateral side of the animals 2 weeks after treatment with MPTP. (B and C) TH-immunopositive (B) or cresyl violet-stained (C) neurons from the ipsilateral or the contralateral region of SNc were quantified. The data are the mean ± SEM (n = 3–4). (D) The expression of Pink1/mutants 1 week after viral delivery of GFP vector (lane 1), WT Pink1 (lane 2), ΔNPink1 (lane 3), G309D (lane 4), and K219M (lane 5) to mouse brain and protein are processed for immunoblotting. The asterisk denotes significance (P < 0.05) when compared with GFP control.

Fig. 5.

Subcellular distribution of Pink1 in HEK293, NIH 3T3 cells, and cortical neurons. (A, B, D) Supernatants collected after centrifugation at 1,000 × g of cell lysate from WT Pink1 transfected (A), untransfected (B), HEK293, or NIH 3T3 (D) cells were subjected to sequential centrifugation, and pellets were analyzed by SDS/PAGE and probed by antibodies to either Pink1 (A, B, D) or to various organelle markers as indicated (C, D). 3k, nuclei, heavy mitochondria, and plasma membrane; 10k, mitochondria, lysosomes, and Golgi; 20k, lysosomes, large vesicles, and rER; 100k, ER vesicles, plasma membrane, Golgi, and endosomes; 100k sup, soluble cytoplasmic proteins. (D) Alternatively, neurons were fractionated into cytoplasmic and mitochondrial fraction in a one-step differential centrifugation as noted in Materials and Methods. This procedure was performed because of the limited neuronal extract available relative to that obtained from cultured fibroblasts.